DESCRIPTION The overall goal of this project is to contribute to the understanding of the role of ion channel diversity in the central nervous system (CNS). The focus is on K+ channels, which underlie many of the differences in intrinsic electrical properties that characterize specific neurons, thus contributing to the complexity of neuronal information coding. Modulation of K+ channel function is one of the key mechanisms by which neurotansmitters and other stimuli, affect excitability. K+ channel genes have already been found to be responsible for two human genetic diseases. This project analyzes the expression and function in the CNS of a family of K+ channels genes in mammals known as KV3, ShIII, or Shaw-related genes. Genes in this family are being considered as causes for human genetic disease, including and episodic choreoathetosis and deafness of central origin. The emphasis is in Kv3.2, a Kv3-related gene which encodes several subunits of voltage-gated K+ channels by alternative splicing, and which so far have only been detected in the CNS. The majority of Kv3.2 mRNAs are produced by thalamic relay neurons throughout the thalamus, a structure in the brain which functions as a gate between the information received form the outside world and the generation of consciousness in the cerebral cortex, since sensory input must first, with few exception, be processed by thalamic relay neurons, before it can reach the neocortex. It has been recently shown that in thalamic relay neurons, the Kv3.2 subunits are localized mainly in the terminal fields of the axons of these cells, the thalamocortical projections. It has also been demonstrated that in vivo, Kv3.2 channels are modulated by phosorylatation and dephosphorylation meditated by the cAMP and the nitric oxide (NO) and cGMP cascades, respectively. This modulation may underlie changes in synaptic transmission in thalamocortical synapses, and thus be involved in the regulation of global states of awareness such as sleep, arousal, coma or changes in attention. Experiments are proposed to further investigate the roles of Kv3.2 channels in the CNS. The composition and properties of native Kv3.2 channels in neurons are not known, because the components have been cloned in the absence of prior biochemical analysis. A combined biochemical, electrosphsiological, and gene-elimination approach is proposed to characterize native Kv3.2 channels, including elucidating the subunit composition of native Kv3.2 proteins and the electrophysiological characterization of native Kv3.2 channels in neurons that express them somatically. To understand the different functional contributions of Kv3.2 and other Kv3 channels, the subsets of neurons expression Kv3.2 proteins in the cortex and the hippocampus will be identified utilizing double-labeling techniques. To gain further insights into the roles of phosphorylation of Kv3.2 proteins, their precise localization in thalamocortical terminal will be compared to that of the components of the cAMP, cGMP, and NO cascades, and experiments will be performed to discover neurotransmitters and neuropeptides that regulate the phosphorylation of Kv3.2 channels in pinched cortical presynaptic terminals.